Universität zu Köln

Reusch, Björn ORCID: 0000-0002-8963-6835 (2023). MAGED2: the gene associated with Bartter syndrome type 5 is a key regulator of Gαs signalling in the developing kidney. PhD thesis, Universität zu Köln.

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Abstract

Pathogenic variants in the Melanoma-Associated Antigen D2 (MAGED2) gene have been identified as the cause of the severe but transient antenatal Bartter syndrome type 5 (BS5) accounting for approximately 10% of all Bartter syndrome cases. The disorder is characterized by severe polyhydramnios leading to premature delivery and still birth as well as postnatal polyuria that declines after a few weeks or months followed by normal further development. The underlying molecular mechanisms of BS5 and especially the function of MAGED2 in kidney physiology had been largely unknown so far. In this study, we show that Maged2 expression in mouse embryonic kidneys declines during development. As MAGED2 interacts with G-protein alpha subunit Gαs (GNAS), it was inferred to play a role in G-protein coupled receptor (GPCR) signalling. Accordingly, we showed that knockdown of MAGED2 in HEK293T cells induces major changes in protein phosphorylation but not in protein abundance. In the murine collecting duct cell line mpkCCD, Maged2 knockdown modulated vasopressin type 2 receptor (V2R)-induced phosphorylation-dependent signalling in terms of cAMP kinetics and weakened phosphorylation on downstream targets like cAMP response element-binding protein (CREB). Unexpectedly, Maged2 knockdown resulted in vitro in a marked increase of the water channel aquaporin-2 (AQP2) abundance upon long-term V2R activation, which was mediated transcriptionally. To further analyse Maged2 function in vivo, we generated a knock-in mouse model of the human mutation p.R446C via CRISPR/Cas9. Mendelian ratio of Maged2R446C/Y mice was clearly skewed towards the wildtype genotype. Affected male mice also showed impaired embryonic development in form of general paleness as well as slightly decreased body and kidney weight. On the proteome level MAGED2 loss in P0 kidneys led to significant alterations of abundance of 307 proteins, with 112 proteins showing increased and 195 proteins showing decreased expression. Remarkably, Gαs was significantly upregulated, while the Thiazide-Sensitive Sodium-Chloride Cotransporter (NCC) was significantly downregulated. Localization studies in kidneys from P0 Maged2R446C/Y mice could not show an aberrant localization of targets defective in other types of BS like the cotransporters Na-K-2Cl Cotransporter 2 (NKCC2), NCC and the Potassium Inwardly Rectifying Channel Subfamily J Member 1 (ROMK), while the apical localization of AQP2 and also its phosphorylated form pAQP2 were clearly impaired. A similar diffuse instead of apical AQP2 distribution was observed in the only available kidney sections from a stillborn BS5 infant. Taken together, MAGED2 modulates GPCR-signalling at a specific period of time and acts as a desensitizer of V2R in vitro. Moreover, we demonstrate that Maged2 loss in vivo impairs the abundance and the targeting of numerous renal proteins which could explain the overall severe character of the disease in comparison to other forms of Bartter syndrome. The studies presented here serve as a basis for further research on BS5, potentially allowing the development of targeted treatments for this disease. Especially the mouse model will help us to dissect the complex effects of MAGED2 in health and disease and integrate the discovery in the context of tubulopathies and general fluid homeostasis. Ultimately, backtracking the reductive cascade of MADED2 functions will yield a more precise understanding of fluid and electrolyte homeostasis during embryonic development and beyond.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Reusch, Björn bjoern.reusch@uk-koeln.de orcid.org/0000-0002-8963-6835 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-655764
Date:	2023
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Zentrum für Molekulare Medizin
Subjects:	Life sciences Medical sciences Medicine
Uncontrolled Keywords:	Keywords Language Bartter syndrome English MAGED2 English GPCR English Proteomics English Mouse model English
Date of oral exam:	24 April 2023
Referee:	Name Academic Title Wirth, Brunhilde Prof. Dr. Gehring, Niels Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/65576